Propylisopropyl acetic acid and propylisopropyl acetamide stereoisomers, a method for their synthesis and pharmaceutical compositions containing them

ABSTRACT

The present invention relates to racemic propylisopropyl acetic acid and propylisopropyl acetamide and their isomers in their racemic and stereospecific forms, for use in treatment of neurological and psychotic disorders, and affective disorders and to treat pain, headaches and migraines. The isomers are of the compound formula I  
                 
 
     R 1  is a methyl or ethyl group;  
     R 2  is H, methyl or an ethyl group;  
     R 3  is ethyl or a propyl group; and  
     R 4  is a hydroxyl or amide group,  
     and the total number of carbon atoms in said compound is 8, provided that when R1 is a methyl group and R4 is an amide group, R2 and R3 are not ethyl, further provided that when R1 is an ethyl and R4 a hydroxyl group, only stereoisomers of the compound are referred to.  
     The present invention further relates to a method for the stereoselective synthesis of the 2R stereoisomer of PID and PIA. The present invention also relates to pharmaceutical compositions containing as an active ingredient a racemic mixture or stereoisomers of the compounds of the general formula (I), which are useful for the treatment of neurological and psychotic disorders, and affective disorders and to treat pain, headaches and migraines.

FIELD OF THE INVENTION

[0001] The present invention generally relates to propylisopropyl aceticacid (PIA) and propylisopropyl acetamide (PID) in their racemic andstereospecific forms, to some of their isomers and to stereoisomersthereof, for use in treatment of neurological and psychotic disorders,and affective disorders and to treat pain, including headaches andmigraine pains. The present invention further relates to a method forthe synthesis of PIA and PID stereoisomers. The present inventionfurther relates to pharmaceutical compositions containing, as an activeingredient, said racemic or stereoisomer forms.

BACKGROUND OF THE INVENTION

[0002] Headaches, especially in the form of migraine pain are a widespread malady.

[0003] Valproic acid (VPA), also used in antiepileptic therapy, is adrug which was approved for the treatment of migraine and has beenutilized in the treatment of epilepsy for the last 25 years with a fewside effects. Two major side effects being teratogenicity andhepatotoxicity, have been associated with valproate therapy.

[0004] In humans, valpromide (VPD), which is also used as ananticonvulsant agent, is a prodrug of valproic acid (VPA). It was foundto be more potent than VPA though it exerted more significant sedativeside effects (Loscher W. and Nau H. (1985) Neuropharmacology 24:427-435).

[0005] Isomers of VPD, such as valnoctamide (VCD-valmethamide or2-ethyl-3-methyl pentanamide), were found to be more potent thanvalproic acid as anticonvulsants (Haj-Yehia A. and Bialer M. (1989)Pharm Res 6:683-9). Stereoselectivity has been shown in pharmacokineticsin man for an amide of an aliphatic short-chain fatty acid such asvalnoctamide (VCD) (Barel S., Yagen B., Schurig V., Soback S., PisaniF., Perucca E. and Bialer M. (1997) Clin. Pharmacol. and Therap. 61 (4):442-449). This work demonstrated that VCD pharmacokinetics (PK) inhumans is stereoselective, with one isomer exhibiting a much higherclearance and a shorter half-life compared with the other threestereoisomers.

[0006] The present invention relates to the stereoisomers of PIA and ofPID and to their isomers (such as VPD and VCA) for use in treatment ofneurological and psychotic disorders, and affective disorders and totreat pain, such as head aches. Although VPA and VPD analogues (such asVCA and VCD) were implicated in the treatment of epilepsy, there is noevidence that they in their racemic form or their individualstereoisomers are active in the treatment of neurological and psychoticdisorders, affective disorders and pain.

SUMMARY OF THE INVENTION

[0007] The present invention relates to racemic propylisopropyl aceticacid and propylisopropyl acetamide and their isomers in their racemicand stereospecific forms, for use in treatment of neurological andpsychotic disorders, and affective disorders and to treat pain,headaches and migraines, wherein the isomers are of the compound formulaI

[0008] wherein

[0009] R₁ is a methyl or ethyl group;

[0010] R₂ is H, methyl or an ethyl group;

[0011] R₃ is ethyl or a propyl group; and

[0012] R₄ is a hydroxyl or amide group,

[0013] wherein the total number of carbon atoms in said compound is 8,provided that when R1 is a methyl group and R4 is an amide group, R2 andR3 are not ethyl, further provided that when R1 is an ethyl and R4 ahydroxyl group, only stereoisomers of the compound are referred to,further provided that when the compounds described by formula I arevalpromide (R₁=ethyl, R₂=H, R₃=propyl, R₄=NH₂), di-isopropylacetic acid(R₁=methyl, R₂=methyl, R₃=isopropyl, R₄=OH) and valnoctamide (R₁=ethyl,R₂=methyl, R₃=ethyl, R₄=NH₂), these compounds are excluded.

[0014] The present invention further relates to a method for thestereoselective synthesis of the 2R stereoisomer of PID and PIAcomprising;

[0015] (a) synthesizing (4S)-3-(1′-oxopentyl)-4-benzyl-2-oxazolidinonefrom (4S)-benzyl-2-oxazolidinone (or other related oxazolidinoneauxiliaries) and valeroyl chloride;

[0016] (b) synthesizing of Isopropyl trifluoromethane sulfonate(isopropyl triflate);

[0017] (c) synthesizing(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone

[0018] (d) synthesizing (2R)-propylisopropyl acetic acid ((2R-PIA) andsubsequently;

[0019] (e) synthesis of (2R)-propylisopropyl acetamide.

[0020] and to a method for the stereoselective synthesis of the 2Sstereoisomer of PID and PIA comprising;

[0021] (a) Synthesizing(4R,5S)-3-(1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone from(4R,5S)-4-methyl-5-phenyl-2-oxazolidinone (or other relatedoxazolidinone auxiliaries) and valeroyl chloride;

[0022] (b) synthesizing(4R,5S,2′S)-3-(2′-isopropyl-1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone;

[0023] (c) synthesizing(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone;

[0024] (d) synthesizing (2S)-propylisopropyl acetic acid ((2S)-PIA)andsubsequently;

[0025] (e) synthesis of (2S)-propylisopropyl acetamide.

[0026] The present invention also relates to pharmaceutical compositionscontaining as an active ingredient a racemic mixture or stereoisomers ofthe compounds of the general formula (I), which are useful for thetreatment of neurological and psychotic disorders, and affectivedisorders and to treat pain, headaches and migraines.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention generally relates to propylisopropyl aceticacid (PIA) and propylisopropyl acetamide (PID) in their racemic andstereospecific forms, to some of their isomers and to stereoisomersthereof, for use in treatment of neurological and psychotic disorders,and affective disorders and to treat pain, including head aches andmigraine pains. The present invention further relates to the 2S and 2RPIA and PID stereoisomers and to a method for their synthesis. Thepresent invention also relates to pharmaceutical compositionscontaining, as an active ingredient, these racemic mixtures orstereoisomers.

[0028] The present invention relates to PID and PIA or PIA isomers, suchas valnoctic acid (VCA) or PID isomers, such as valpromide (VPD) intheir racemic or stereospecific forms and to the stereoisomers of chiralvalproyl amide analogous of valproic acid (VPA) such as PID, which maybe useful in the treatment of neurological and psychotic disorders, andaffective disorders and to treat pain.

[0029] The present invention relates to pharmaceutical compositionscontaining, as their active ingredient, PIA or PID isomers of thegeneral formula (I):

[0030] wherein

[0031] R₁ is a methyl or ethyl group;

[0032] R₂ is H, methyl or an ethyl group;

[0033] R₃ is ethyl or a propyl group; and

[0034] R₄ is a hydroxyl or amide group,

[0035] and wherein the total number of carbon atoms is 8.

[0036] provided that when R1 is a methyl and R4 is an amide group, R2 isnot an ethyl and R3 is not an ethyl, further provided that when R1 is anethyl and R4 a hydroxyl group, only stereoisomers of the compound arereferred to, further provided that when the compounds described byformula I are valpromide (R₁=ethyl, R₂=H, R₃=propyl, R₄=NH₂),di-isopropylacetic acid (R₁=methyl, R₂=methyl, R₃=isopropyl, R₄=OH) andvalnoctamide (R₁=ethyl, R₂=methyl, R₃=ethyl, R₄=NH₂), these compoundsare excluded.

[0037] For example, in PIA R1 is a methyl, R2 is a methyl, R3 is apropyl and R4 is a hydroxyl and in PID R1 is a methyl, R2 is a methyl,R3 is a propyl and R4 is an amide group.

[0038] The present invention further relates to a method for thestereoselective synthesis of the propylisopropyl acetic acid andcorresponding amide (PIA and PID)) stereoisomers (2R and 2S).

[0039] The method of synthesis of the 2R stereoisomers, which isdescribed in FIGS. 1 and 2, comprises the following steps;

[0040] (a) synthesizing (4S)-3-(1′-oxopentyl)-4-benzyl-2-oxazolidinone(1) from (4S)-benzyl-2-oxazolidinone (or other related oxazolidinoneauxiliaries) and valeroyl chloride;

[0041] (b) synthesizing of Isopropyl trifluoromethane sulfonate(isopropyl triflate) (2);

[0042] (c) synthesizing(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone(3);

[0043] (d) synthesizing (2R)-propylisopropyl acetic acid (2R-PIA) (4)and subsequently;

[0044] (e) synthesis of (2R)-propylisopropyl acetamide (5).

[0045] The method of synthesis of the 2S stereoisomers, which isdescribed in FIG. 3, comprises the following steps;

[0046] (a) Synthesizing(4R,5S)-3-(1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone (6) from(4R,5S)-4-methyl-5-phenyl-2-oxazolidinone (or other relatedoxazolidinone auxiliaries) and valeroyl chloride;

[0047] (b) synthesizing(4R,5S,2′S)-3-(2′-isopropyl-1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone(7);

[0048] (c) synthesizing(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone

[0049] (d) synthesizing (2S)-propylisopropyl acetic acid ((2S)-PIA) (8)and subsequently;

[0050] (e) synthesis of (2S)-propylisopropyl acetamide ((2S)-PIA) (9).

[0051] The said invention will be further illustrated by the followingexperiments. These experiments do not intend to limit the scope of theinvention, but to demonstrate and clarify it only.

[0052] The two enantiomers of PID, (2S)-PID and (2R)-PID, were tested inmice and rats for their antiepileptic (anticonvulsant) activity and forneurotoxicity. Following i.p. administration to mice and oraladministration to rats, (2R)-PID was more active and showed betterpotency than (2S)-PID in the MES and sc Met tests.

[0053] In dogs, following iv administration, (2R)-PID had a lowerclearance and longer half-life than (2s)-PID, a fact that may contributeto the better anticonvulsant activity of (2R)-PID. The betteranticonvulsant activity (compare to VPA) of PID and the lack of itsteratogenicity, increases the likelihood that other CNS activitiesexerted by VPA (treatment of neurological and psychotic disorders,affective disorders pain) is more pronounced by PID in its racemic andstereoisomers forms.

[0054] The method for the asymmetric synthesis of the 2R and 2S PID andPIA stereoisomers will be further described by the following examples:

[0055] Synthesis of (2R)-propylisopropyl acetamide

[0056] 1. (4S)-3-(1′-oxopentyl)4-benzyl-2-oxazolidinone

[0057] Under N₂ to a cooled solution (−78° C.) of(4S)-benzyl-2-oxazolidinone (25 g) in dry THF (150 ml) was addeddropwise a solution of n-BuLi (97 ml, 1.6 M in hexane). After stirringthe reaction mixture for 30 min, valeroyl chloride (20.1 ml) was addeddropwise via cannula, the reaction was slowly warmed to 0° C., at thistemperature stirred for 2.5 hours and quenched by saturated NH₄CLsolution. After evaporation of THF the residue was extracted withdichloromethane (DCM) (3×150 ml). The combined organic extracts washedwith water, saturated brine and dried over MgSO₄. The product (27.6 g)was crystallized from 10% EtOAc in PE, yield 75%.

[0058] 2. Isopropyl trifluoromethane sulfonate (isopropyl triflate)

[0059] Under N₂ to a cooled (−15° C.) solution of dry isopropanol (10.4ml) and dry Et₃N (25.2 ml) in dry DCM (200 ml) was added dropwise acooled (−15° C.) solution of Tf₂O (triflic anhydride^(a)) (49.0 g in 50ml of DCM). The reaction mixture was stirred for an hour then quenchedwith cooled (0° C.) HCl solution (0.25 M, 2×350 ml). The organic phasewas washed with cooled (0° C.) solution of NaHCO₃ (0.5 M, 2×175 ml),dried over MgSO₄ and concentrated. The yellowish liquid obtained wasdissolved in a cooled (0° C.) pentane solution (50 ml), filtered througha short MgSO₄ plug and concentrated. The product (18.3 g) was obtainedin 55% yield, dissolved in dry pentane and kept at −20° C. until used.

[0060] (^(a) triflic anhydride is trifluoromethanesulfonic anhydride)

[0061] 3.(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone

[0062] Under N₂ to a cooled (−78° C.) solution of dry diisopropylamine(11 ml) in dry THF (40 ml) was added dropwise n-BuLi (49 ml, 1.6 Msolution in hexane). After stirring the reaction mixture for 30 min acooled (−78° C.) solution of(4S)-3-(1′-oxopentyl)-4-benzyl-2-oxazolidinone (18.5 g in 70 ml dry THF)is slowly added via cannula. After stirring for 1 hour, a cooled (−78°C.) solution of isopropyl triflate (15 g in 40 ml dry THF) is added viacannula. The reaction slowly warmed to −20° C., left overnight at −20°C. and quenched by saturated NH₄Cl solution. Total reaction time was 20hours. THF evaporated and the residue extracted with Et₂O (3×150 ml) anddried over MgSO₄. Purification of the crude product (23 g of a yellowoil) by column chromatography (silica gel, 0.5-3% EtOAc in PE) afforded8.8 g of a yellowish oil, 41% yield.

[0063] 4. (2R)-propylisopropyl acetic acid ((2R)-PIA)

[0064] To a cooled (0° C.) solution of(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone (8.8 g)in a mixture of THF:DDW (4:1, 550 ml) was added H₂O₂ (30%, 19.3 ml)followed by a solution of LiOH (2.44 g in 50 ml DDW). After stirring for3 hours the reaction mixture was warmed to room temperature (˜23° C.)and left overnight. After 24 hours the reaction mixture was cooled to 0°C. quenched by sodium sulfite (21 g in 100 ml DDW) and stirred for anadditional hour. THF evaporated and the basic aqueous phase (PH=11)extracted with DCM (3×100 ml). The chiral auxiliary,(4S)-benzyl-2-oxazolidinone was obtained after evaporation of the DCMand crystallization from 20% EtOAc in PE, 80% yield. The aqueous phasewas then acidified with concentrated HCl (PH=2) and extracted with EtOAc(3×100 ml). The combined organic extracts washed with saturated brine,dried on MgSO₄, evaporated and afforded the product, a colorless oil(3.49 g), yield 83%.

[0065] 5. (2R)-propylisopropyl acetamide ((2R)-PID)

[0066] Under N₂ to a cooled (0° C.) solution of (2R)-PIA (3.3 g)dissolved in dry DCM (100 ml) and dry DMF (1.77 ml) was added dropwise asolution of oxalyl chloride (34.3 ml, 2.0 M solution in DCM). Afterstirring one hour the DCM and excess oxalyl chloride were evaporated byN₂ stream. In order to remove traces of oxalyl chloride the crudeproduct was treated with dry DCM (2×20 ml) which was evaporated by N₂stream. To the crude reaction mixture dissolved in cooled (0° C.) dryDCM (100 ml) was added NH₄OH (20 ml, 25% solution in water) and thereaction mixture stirred for an hour. The organic phase washed withwater and half saturated brine, dried over MgSO₄, filtered andconcentrated. The product was crystallized from 20% EtOAc in PE toafford 2.14 g, 65% yield.

[0067] Synthesis of (2S)-propylisopropyl acetamide

[0068] 1. (4R,5S)-3-(1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone

[0069] (4R,5S)-3-(1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone wassynthesized from (4R,5S)-4-methyl-5-phenyl-2-oxazolidinone and valeroylchloride by the same procedure as(4S)-3-(1′-oxopentyl)-4-benzyl-2-oxazolidinone. The product (29.55 g)was obtained in 84% yield.

[0070] 2.(4R,5S,2′S)-3-(2′-isopropyl-1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone

[0071](4R,5S,2′S)-3-(2′-isopropyl-1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinonewas synthesized from(4R,5S)-3-(1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone and isopropyltriflate by the same procedure as(4S,2′S)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone. Theproduct (5.53 g) was obtained in 32% yield.

[0072] 3. (2S)-propylisopropyl acetic acid ((2S)-PIA)

[0073] (2S)-PIA was synthesized from(4R,5S,2′S)-3-(2′-isopropyl-1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinoneby the same procedure as (2R)-PIA. The product (2.33 g) was obtained in89% yield.

[0074] 4. (2S)-propylisopropyl acetamide ((2S)-PID)

[0075] (2S)-PID was synthesized from (2S)-PIA by the same procedure as(2R)-PID. The product (1.54 g) was obtained in 67% yield.

[0076] Pharmacokinetic Studies

[0077] Pharmacokinetic experiments were carried out on six mongrel dogsweighing 18-25 kg. Dogs were housed in an animal farm and were broughtto the lab repeatedly every two-three weeks for crossover experimentsafter an overnight fast. Each dog was inserted with a urine (Levin'stube, Pennine Healthcare, Derby, UK) and two venous (20G/32 mm Venflon2, Ohmeda, Helsingborg, Sweden) catheters located on different legs.Dogs were fed with commercial dog food four hours after drug injection,and had free access to water during the whole experiment.

[0078] Each dog was intravenously injected with 70 nmole/kg (10 mg/kg)of each enantiomer separately or 140 nmole/kg of the racemate, dissolvedin 2 ml of 96% ethyl alcohol. Venous blood samples (6 ml) were withdrawnvia an indwelling catheter from the other leg than used for injection,and transferred into heparinized tubes. The blood samples werecentrifuged at 3000 g for 10 minutes, plasma was then separated andstored at 20° C. until analyzed. Blood collection commenced at 5minutes, and continued up to 12 hours after injection. Urine sampleswere collected in 1-2 hour intervals beginning at 1 hour and up to 12hours after injection. The urine volume was recorded and an aliquot wasstored at 20° C. until analyzed.

[0079] Assay of plasma samples: to test tubes containing 2 μg of theinternal standard (diisopropyl acetamide), 0.5 ml of plasma (thawed atroom temperature) and 5 ml of tert-butyl methyl ether (TBME) was added.The test tubes were vigorously vortexed for 30 sec and centrifuged at3000 g for 10 minutes. The organic phase was separated and dried underreduced pressure using a vortex evaporator. Samples were thenreconstituted with 150 μl of chloroform and dried under reduced pressurewithout vortexing. The sample was again reconstituted with 30 μl ofchloroform, of which 2 μl were injected into the GC apparatus.

[0080] The column used for the chromatographic analysis of PIDenantiomers was a Mosandl-methyl capillary column (10 m, 0.25 mm, 0.25μm) coated with: Heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin as thestationary phase and nitrogen as carrier gas (takeo carbohydr res).Column head pressure was set at 50 KPa, split ratio 1:30, oventemperature 120° C., injector temperature 250° C. and detectortemperature 250° C. At these conditions S-PID had a retention time of4.6 min, R-PID 5.1 min.

[0081] The results (FIGS. 4 and 5) show-that when enantiomers are givenindividually, PID exhibits a clear stereospecific kinetics. But, whenPID is given in a racemic mixture, no sterospecifity is observed and theparmacokinetic parameters are very close to those of the individual Renantiomer.

[0082] The Pharmacokinetic parameters of the individual enantiomers andof the racemic mixture are summarized in Table 1 and Table 2respectively.

[0083] (CL=clearance, V_(β)=volume of distribution, Vss=V_(β) at steadystate, MRT=Mean Residence Time, E=liver extraction ratio, fe=fractionexcreted unchanged in the urine) TABLE 1 Pharmacokinetic Parameters ofPID Enantiomers in Dogs given as individual enantiomers (10 mg/kg, viaiv) (R)-PID (S)-PID S/R CL (L/h)  6.8 ± 1.6 11.3 ± 1.5 1.66* V_(β) (L)13.1 ± 2.2 17.0 ± 2.9 1.29 Vss (L) 16.0 ± 1.2 19.1 ± 2.7 1.19 t½ (h)1.37 ± 0.2 1.04 ± 0.1 0.76* MRT (h) 2.57 ± 0.5 1.83 ± 0.3 0.71* E (%)15.0 ± 4.6 23.7 ± 3.5 1.58* fe (%) 0.50 ± 0.4 0.40 ± 0.4 0.78*

[0084] TABLE 2 Pharmacokinetic Parameters of PID Enantiomers in Dogsgiven in a racemic mixture (20 mg/kg, via iv) (R)-PID (S)-PID S/R CL(L/h)  6.0 ± 1.7  6.0 ± 1.8 1.0 V_(b) (L) 10.2 ± 0.9 10.4 ± 1.4 1.0 Vss(L) 15.7 ± 1.5 15.7 ± 0.9 1.0 t½ (h) 1.25 ± 0.3 1.27 ± 0.3 1.0 MRT (h)2.83 ± 0.6 2.87 ± 0.7 1.0 E (%) 12.5 ± 4.0 12.1 ± 4.1 1.0 fe (%)  0.60 ±0.45  0.66 ± 0.46 1.1

[0085] Epoxide Hydrolase Inhibition Assay

[0086] PID is known to be an inhibitor for Epoxide Hydrolase (EH)activity. A study was conducted in order to explore the possibility ofPID stereospecificy in the EH inhibition. The study was conducted asfollows:

[0087] The in vitro microsomal EH inhibitory potency of racemic PID andits individual enantiomers was measured in human liver microsomes withS-(+)-styrene oxide (SO) as substrate. Microsomes were prepared fromhunan liver #135 (HL-135), genotipically classified as a wild type EH,according to the previously published procedure (Rattie (1989) DrugMetabolism and Disposition, 17: 265-270). The rate ofS-(+)-1-phenyl-1,2-ethanediol (PED) formation was measured in microsomalincubations, as previously described, with slight modifications (Kerr(1989) Clinical Pharm. Therp. 46: 82-93). Briefly: Racemic PID, S-PID,R-PID or 0.1M sodium phosphate buffer PH 7.4 were preincubated withmicrosomal protein for 1.5 min at 37° C., before the reaction wasstarted by addition of SO. The reaction was terminated by the additionof 3 ml n-hexane, rapid vortexing for 30 sec, and placing the tubes onice until processed further. The background hydrolysis rate for SO wasmeasured by replacement of microsomal protein with an equal amount ofdenatured microsomal protein. All reported PED formation rates have beencorrected for the non-enzymatic hydrolysis. The final proteinconcentration was 5.34 μg/ml. SO was added in a 15 μl acetonitrilesolution, such that the final SO concentration was 25 μM (equals theK_(m) of SO). PID and its individual enantiomers were added in a 15 μlmethanolic solution, such that the final concentration of organicsolvent was 1%. The inhibition reactions were investigated at fiveconcentrations ranging from 4 to 30 μM. Positive control for 50%inhibition was done by using 5 μM VPD (equals the IC₅₀ of VPD). Alldeterminations were made in triplicates.

[0088] PED was extracted and assayed using a reverse phase HPLCprocedure previously published, with slight modifications (Kerr, (1989)Clinical Pharm. Therp. 46: 82-93). Internal standard was felbamate 1.6μg in 100 μl methanolic solution. Microsomal incubations were extractedwith 7 ml TBME. Mobile phase composition: double distilledwater/acetonitrile/methanol 70:20:10. Flow rate was set at 1.2 ml/minand the compounds detected by UV absorption at 210 nm. Chromatographicseparation was carried out on a Zorbax C₈ column (5 μM, 4.6×25 cm)equipped with a C₈ guard column (5 μM, 4.6×1.0 cm).

[0089] The inhibition of EH mediated SO hydrolysis by PID and theindividual enantiomers; S-PID and R-PID, was examined in microsomalsuspensions that were prepared from a single human liver, HL-135. ThePED formation rates are presented inn FIG. 6. From percent remainingactivity of EH vs. inhibitor concentration plots, IC₅₀ values could bederived for each of the tested compounds. Racemic PID had an IC₅₀ of8.55 μM, S-PID an IC₅₀ of 7.60 μM and R-PID an IC₅₀ of 11.20 μM.

[0090] Average non-enzymatic SO hydrolysis rates were 6.67±0.47% of thecontrol enzymatic hydrolysis rates. VPD (5 μM) as a positive controlinhibited SO hydrolysis by 51.5±2.4% of the control enzymatic hydrolysisrates. QC of four different PED concentrations within the PEDconcentration range had an accuracy of 0.55-2.70% and reproducibility (%CV) of 1.49-5.61%.

[0091] Biological Activity

[0092] 1. Teratogenicity Study

[0093] 1a. Teratogenicity (Finnel) Study in SWV Mice

[0094] Teratogenicity was evaluated in the highly inbred SWV mice strainon the basis of their known susceptibility to VPA-induced NTDs (Finnelet al. (1988) Teratology 38: 313-320). The mice were maintained on a 12h light cycle in the Animal Resources Facility at The college ofVeterinary Medicine, Texas A&M University. Mice were pathogen free andwere allowed free access to Wayne TekLad rodent chow and tap water.Virgin females, 40-60 days of age were bred overnight and examined thenext morning for the presence of vaginal plugs. The beginning ofgestation (day 0) was set at 10 P.M. of the previous evening, themidpoint of the dark cycle (Finnel et al. (1988) Teratology 38:313-320). Ten dams were randomly assigned to each of the testedcompounds; racemic PID, S-PID and R-PID. At day 8.5 of gestation, eachdam was exposed to a single intraperitoneal (ip) injection of the testedcompound (500-600 mg/kg) or the vehicle (1% carboxymethylcellulose-CMC). Following administration, the dams were returned totheir cages until day 18.5 of gestation. At that time the dams weresacrificed by cervical dislocation, the abdomen opened and the uterinecontents removed. The location of all-viable embryos or fetuses andresorption sites were recorded, and the embryos were examined for thepresence of exencephaly.

[0095] Teratogenicity in the SWV mice strain was evaluated following asingle ip administration of PID, S-PID and R-PID to ten dams at day 8.5of gestation. Teratogenic data is presented in Table 1. Both PID andR-PID were administered at doses of 600 mg/kg. Due to several incidencesof maternal lethality following 600 mg/kg administration of S-PID,teratogenicity was further investigated at 500 mg/kg. Both racemic andR-PID failed to induce exencephaly in the SWV embryos. S-PID caused 0.8%exencephaly, but this was not different from controls (0%). Resorptionrates induced by PID, S-PID and R-PID were 6.3%, 6.1% and 10.4%,respectively, which is not different from controls, 8.6%. TABLE 3Teratogenic effects of PID, R-PID and S-PID in SWV mice Dose Compound(mg/kg) Litters Implants Resorptior Live Fetuses Exencephaly Control³  011 148  1 (0.7) 147 0 PID 600 10 126  8 (6.3)* 118 0 R-PID 600 10 134 14(10.4) 120 0 S-PID 500 10 131  8 (6.1)* 123 1 (0.8)

[0096] 1b. Teratogenicity (Nau) Study in Mice of NMRI Strain

[0097] Mice of the NMRI strain (Harlan-Winkelmann GmbH, 33176 Borchen,Germany) were kept under controlled conditions: Room temperature (21±1°C.), relative humidity (50±5%), and a 12 hour light-dark cycle with thelight period from 10 a.m. to 10 p.m. Females weighing 28 to 36 g weremated with males of the same strain for 3 hours (from 6 a.m. to 9 a.m.).Animals with vaginal plugs were separated, and the following 24 hourperiod was designated as day 0 of pregnancy. The animals were given freeaccess to food (Altromin 1324 diet, Lage, Germany) and tap water.Approval for the study was obtained from the Department of Health.

[0098] Sodium valproate (VPA-Na), racemic PID, (2R)-PID and (2S)-PIDwere suspended in a 25% Cremophor EL aqueous solution. For anothertreatment group, VPA-Na was dissolved in distilled water. The pregnantdams were injected with a single 3 mmol/kg subcutaneously dose (10 ml/kgvolume administered) on the morning of day 8 of gestation. Mice of thecontrol group were injected with the vehicle, 25% Cremophor EL solution(10 ml/kg volume administered). On day 18 of gestation the dams weresacrificed by cervical dislocation, the uteri removed and the number ofimplantations, resorptions and dead fetuses recorded. Living fetuseswere weighed individually and inspected for the presence of externalmalformations.

[0099] Teratogenic potency of racemic PID and the individual enantiomerswas evaluated in NMRI mice following a single 3 mmol/kg subcutaneousinjection to pregnant dams at day 8 of gestation, as presented in Table4. Racemic PID and the individual enantiomers failed to induceexencephaly in the developing mouse embryos, whereas VPA caused 37% and73% exencephaly in living fetuses when administered in aqueous solutionand Cremophor suspension, respectively. Fetal deaths and earlyresorptions expressed as embryolethality was significantly increased inVPA-treated animals, whereas all PID groups and controls had comparablerates. All treatment groups (PID and VPA) had significant reduction infetal weight. TABLE 4 Teratogenicity of racemic PID, (2R)-PID and(2S)-PID in NMRI mice at day 18 of gestation. Total Live Fetal Embryo-Exence- Dose Litters Implants Fetuses Weight¹ lethality² phaly³ Compoundmmol/kg n n n g n (%) n (%) (2R)-PID 3  9  92  78 1.16 ± 0.08* 14 (15.2) 0 (0) 4 (2S)-PID 3  5  64  58 1.15 ± 0.09*  6 (9.4)  0 (0) 4 Racemic 3 8  96  87 1.21 ± 0.07  9 (9.4)  0 (0) PID4 VPA-Na4 3  7  86  41 0.96 ±0.08* 45 (52.3)** 30 (73.2)** VPA-Na5 3  8  92  63 1.09 ± 0.11* 29(31.5)** 23 (36.5)** Controls — 20 258 238 1.23 ± 0.09 20 (7.8)  3 (1.3)

[0100] 2. Anti-Migraine Activity of PID.

[0101] The GABA transaminase inhibitor and activator, of glutamic acid,the decarboxylase, valproic acid is being used for the treatment ofmigraine. In this study, a valproyl amide analog (PID) in a racemicform, was tested in animal models (rats) for the treatments of migraineand pain, in comparison to VPA.

[0102] The animal model used in this study is the one developed byMoskowitz et al. (F. M. Cutrer, V. Limmroth and M. A. Moskowitz:Possible mechanisms of valproate in migraine prophylaxis, Cephalalgia17:93-100 (1997)). Moskowitz et. al. examined the plasma proteinextravasation following electrical trigeminal ganglion stimulation orintravenous administration of substance P. It was concluded that in thismodel valproic acid blocks plasma extravasation in the meninges throughGABA_(A)-mediated postjunctional receptors probably within the meninges.The dosages required are comparable to those used clinically. Thus,agonists and modulators at the GABA_(A) receptor may become useful forthe development of selective drugs for migraine and cluster headache (W.S. Lee et. al.: Peripheral GABAA receptor mediated effects of sodiumvalproate on durnal plasma protein extravasation to substance P andtrigeminal stimulation, Toward Migraine 2000, F. C. Rose Ed. Elsevier,Amsterdam, 1996, pp.289-319).

[0103] We tested the effects of PID on dural plasma protein (BovineSerum Albumine—BSA) extravasation evoked by unilateral trigeminalgangelion stimulation in anaesthetized rats. The results of this study,shown in FIG. 7, show that PID has inhibitory effects on the duralplasma protein extravasation, i.e., it has the potency for anti-migraineand anti-pain activity.

[0104] 3. Anticonvulsant Activity and Neurotoxicity of PID in Mice

[0105] Individual enantiomers of PID were screened in mice for theiranticonvulsant activity (by the NIH Epilepsy Branch) followingintraperitoneal administration to mice by employing a screeningprocedure which involves: (i) the maximal electoshock (MES) test, whichmeasures seizure spread; (ii) the subcutaneous pentylenetetrazol testsc. Met. Test), which measures seizure threshold; and (iii) the rotorodataxia test, which assesses neurotoxicity.

[0106] Table 6 shows the results obtained in this study: TABLE 6Anticonvulsant Activity and Neurotoxicity of PID in Mice(intraperitoneal administration) PID (S)-PID (R)-PID S/R MES 122 145 1101.32 sc Met 77 80 67 1.19 Neurotox. <120 118 <145 >0.81 PI-MES <0.980.81 <1.3 PI-sc Met <1.56 1.46 <2.2

[0107] 4. Anticonvulsant Activity and Neurotoxicity of PID in Rats

[0108] Individual enantiomers of PID were screened in rats for theiranticonvulsant activity following oral feeding, by the proceduredescribed in the previous section. The ED₅₀ values obtained in thisstudy are as follows:

[0109] ED₅₀ for (2R)—PID: 16 mg/kg

[0110] ED₅₀ for (2S)—PID: 26 mg/kg

[0111] ED₅₀ for racemate: 22 mg/kg

1) Racemic propylisopropyl acetic acid and propylisopropyl acetamide andtheir isomers in their racemic and stereospecific forms, for use intreatment of neurological and psychotic disorders, and affectivedisorders and to treat pain, headaches and migraine, wherein the isomersare of the compound formula I

wherein R₁ is a methyl or ethyl group; R₂ is H, methyl or an ethylgroup; R₃ is ethyl or a propyl group; and R₄ is a hydroxyl or amidegroup, wherein the total number of carbon atoms in said compound is 8,provided that when R1 is a methyl group and R4 is an amide group, R2 andR3 are not ethyl, further provided that when R1 is an ethyl and R4 ahydroxyl group, only stereoisomers of the compound are referred to,further provided that when the compounds described by formula I arevalpromide (R₁=ethyl, R₂=H, R₃=propyl, R₄=NH₂), di-isopropylacetic acid(R₁=methyl, R₂=methyl, R₃=isopropyl, R₄=OH) and valnoctamide (R₁=ethyl,R₂=methyl, R₃=ethyl, R₄=NH₂), these compounds are excluded. 2) A methodfor the stereoselective synthesis of the 2R stereoisomer ofpropylisopropyl acetic acid and propylisopropyl acetamide comprising;(a) synthesizing (4S)-3-(1′-oxopentyl)-4-benzyl-2-oxazolidinone from(4S)-benzyl-2-oxazolidinone, or other oxazolidinone auxiliaries, andvaleroyl chloride; (b) synthesizing of Isopropyl trifluoromethanesulfonate (isopropyl triflate); (c) synthesizing(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone (d)synthesizing (2R)-propylisopropyl acetic acid ((2R-PIA) andsubsequently; (e) synthesis of (2R)-propylisopropyl acetamide. 3) Amethod for the stereoselective synthesis of the 2S stereoisomer ofpropylisopropyl acetic acid and propylisopropyl acetamide comprising;(a) synthesizing(4R,5S)-3-(1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone from(4R,5S)-4-methyl-5-phenyl-2-oxazolidinone, or other oxazolidinoneauxiliaries, and valeroyl chloride; (b) synthesizing(4R,5S,2′S)-3-(2′-isopropyl-1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone;(c) synthesizing(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone(4S,2′R)-3-(2′-isopropyl-1′-oxopentyl)-4-benzyl-2-oxazolidinone;(d) synthesizing (2S)-propylisopropyl acetic acid ((2S)-PIA)andsubsequently; (e) synthesis of (2S)-propylisopropyl acetamide. 4) Amethod for the stereoselective synthesis of the 2R stereoisomer ofpropylisopropyl acetic acid and propylisopropyl acetamide according toclaim 2 wherein step (a) comprises adding n-BuLi to a solution of(4S)-benzyl-2-oxazolidinone at approximately −78° C., stirring andadding valeroyl chloride, warming to approximately 0° C., stirring andquenching obtaining compound (1) and wherein step (b) comprises addingto a solution of isopropanol and Et3N a solution of triflic anhydride atapproximately −15° C., stirring, quenching, obtaining compound (2) in anorganic phase; and wherein step (c) comprises adding n-BuLi to asolution of diisopropylamine oxazolidinone at approximately −78° C.,stirring and adding a solution of (4S)-3-(1′-oxopentyl)-4-benzyl-2-oxazolidinone, stirring and adding a solution of isopropyltriflate, warming the solution to −20° C. and quenching obtainingcompound (3); and wherein step (d) comprises adding H₂O₂ followed by asolution of LiOH to compound (3), stirring and warming to about 23° C.,cooling to 0° C. and quenching obtaining a basic aqueous phase, furtherextracting with dichloromethane, obtaining (4S)-benzyl-2-oxazolidinone,further acidifying said (4S)-benzyl-2-oxazolidinone obtaining compound(4); and wherein step (e) comprises adding a solution of oxalyl chlorideto a solution of compound (4) at approximately 0° C., stirring andadding NH₄OH, stirring and obtaining compound (5) in an organic phase.5) A method for the stereoselective synthesis of the 2S stereoisomer ofpropylisopropyl acetic acid and propylisopropyl acetamide according toclaim 3 wherein step (a) comprises adding n-BuLi to a solution of(4R,5S)-4-methyl-5-phenyl-2-oxazolidinone) at approximately −78° C.,stirring and adding valeroyl chloride, warming to 0° C., stirring andquenching obtaining compound (6); and wherein step (b) comprises addingto a cooled solution of isopropanol and Et3N a solution of of triflicanhydride at approximately −15° C., stirring, quenching, obtainingcompound (2) in an organic phase; and step (c) comprises adding n-BuLito a solution of diisopropylamine at approximately −78° C., stirring andadding a solution of (4R,5S)-3-(1′-oxopentyl)-4-methyl-5-phenyl-2-oxazolidinone, stirring andadding a solution of isopropyl triflate, warming the solution to −20° C.and quenching obtaining compound (7); and wherein step (d) comprisesadding H₂O₂ followed by a solution of LiOH to compound (7), stirring andwarming to about 23° C., cooling to 0° C. and quenching obtaining abasic aqueous phase, further extracting with dichloromethane, acidifyingsaid extract obtaining compound (8); and wherein step (e) comprisesadding a solution of oxalyl chloride to a solution of compound (4) atapproximately 0° C., stirring and adding NH₄OH, stirring and obtainingcompound (9) in an organic phase. 6) Pharmaceutical compositionscontaining as an active ingredient a racemic mixture or stereoisomers ofthe compounds, as defined in claim
 1. 7) Pharmaceutical compositionsaccording to claim 6 wherein the compositions are useful for thetreatment of neurological and psychotic disorders, and affectivedisorders and to treat pain, headaches and migraines.